Although disulfide bonds are critical to the structure of many secreted proteins, and to the regulation of a range of biochemical processes, their biosynthesis in multicellular organisms remains surprisingly cryptic. This application deals with several evolutionarily-related FAD-dependent sulfhydryl oxidases: members of the Quiescin-sulfhydryl oxidase (QSOX) family of flavoproteins, and a representative of the smaller single-domain Erv-like oxidases, augmenter of liver regeneration (ALR). The QSOX enzymes introduce disulfide bonds directly into unfolded reduced proteins, but have also been identified as growth factors in vertebrates (e.g. bone-derived growth factor, placental-derived prostrate growth factor, and erythroid cell stimulating factor). QSOX1 is strongly up-regulated in a number of human cancers (most notably of prostrate and pancreas) and may be involved in the remodeling of the extracellular matrix. ALR shares the same FAD-binding domain as QSOX and is found in a long form (lfALR) in the intermembrane space of the mitochondrion and in a short form (sfALR) functioning in a variety of cellular and extracellular locales. The first of three specific aims of this application explores the molecular mechanism by which two diverse QSOX enzymes (human QSOX1 and the simpler QSOX from the protozoan parasite Trypanosoma brucei) catalyze the efficient oxidation of unfolded reduced protein substrates. The second aim is to search for inhibitors of these enzymes by quantitative high-throughput screening and to continue the design of arsenical inhibitors targeting CxxC motifs in biology. The third aim deals with short and long forms of ALR. We will extend our crystallographic investigations of sf- and lfALR and probe the reductive and oxidative halves of lfALR catalysis by rapid reaction techniques. Finally, we intend to reconstitute oxidative protein folding pathways driven by lfALR and examine their kinetic competence in vitro. Overall, these three aims will contribute to a better understanding of the redox-enzymology of oxidative protein folding in higher eukaryotes.